The entry of viruses into their host cells is a key step in the virus infection pathway, and a potential point for therapeutic intervention. The enveloped alphavirus, Semliki Forest virus (SFV), infects cells via endocytosis followed by a membrane fusion reaction triggered by the acid pH present in intracellular vacuoles. The alphavirus family is comprised of 26 related viruses, some of which are significant pathogens of humans or domestic animals. Major elements of the endocytic infection pathway first described for SFV are also used by a number of other virus families that include important human and veterinary pathogens. A crucial issue in studies of all of these viruses is the molecular mechanism of membrane fusion, a critical function for both viruses and cells. Our goal is to define the molecular features of a membrane fusion reaction, using the well defined SFV system and a combination of biochemical, genetic, and immunological approaches. SFV fusion is mediated by the heterotrimeric viral spike protein, which undergoes an ordered series of conformational changes following exposure to acid pH that culminate in membrane fusion. These conformational changes will be localized by determining the binding sites for a series of monoclonal antibodies specific for the acid form of the spike protein. Binding sites will be defined by competition assays, by identifying the amino acids that comprise the antibody epitopes, and by functional assays of the effects of antibodies in virus fusion. The role of specific spike protein domains in fusion will be evaluated by analysis of our previously obtained virus and spike protein fusion mutants. The mechanisms by which these mutations affect fusion will be determined by analysis of the series of known molecular events that lead to fusion, including acid-dependent conformational changes and interactions with the target membrane. An infectious SFV clone will be used to analyze the effects of the spike protein mutations on virus assembly, infectivity, and fusion. Revertants of a mutation that blocks virus fusion will be selected and characterized for their genotype and fusion mechanism. The SFV E1 spike protein subunit interacts with the target membrane to trigger fusion. A proteolytically truncated form of E1 will be used to analyze the biochemical nature of E1's interaction with the membrane and its requirement for specific lipids, and to identify and characterize the E1 domain involved in membrane binding. A similar but genetically truncated form of E1 will be prepared and assayed for functional activity and suitability for structural studies.